Electrotransformation of Clostridium pasteurianum
By this invention, for the first time, a method for high-efficiency genetic transformation of the anaerobic bacterium Clostridium pasteurianum is provided. Clostridium pasteurianum is a bacterium of substantial industrial importance, due to its selectivity and high productivity of the biofuel and biochemical n-butanol, and its ability to grow on a wide variety of inexpensive substrates. Notable among the substrates that it can utilize as a sole source of carbon and energy is glycerine, which is produced in increasing quantities globally as a by-product of biodiesel processing. The industrial exploitation of Clostridium pasteurianum has previously been impeded by the lack of genetic engineering tools for this bacterium. This invention provides such tools for the first time. Included in the invention is a means for protecting newly introduced DNA from degradation by a restriction enzyme within C. pasteurianum. Then, a detailed protocol is given, which enables high-efficiency transformation of C. pasteurianum via a series of treatments and electroporation conditions which successfully negotiate the resistant cell wall of C. pasteurianum. Finally, the invention discloses selection markers and vector components, which round out the tools required to successfully perform genetic engineering in C. pasteurianum for the first time.
The present invention is directed to bacterial cells and methods for introducing nucleic acids into bacterial cells, and methods and nucleic acids related thereto.
BACKGROUNDBiofuels are regarded as offering a sustainable and environmentally positive replacement for some fossil fuels. Typically, the problem with biofuels is not their performance, but their cost. Most biofuels, in the absence of government subsidies, are more expensive than their fossil fuel counterparts. Thus, reducing the cost of biofuels remains one of the biggest priorities in the biofuel industry. In an analysis of biofuel costs, the cost of the raw feedstock from which the biofuels is produced, is normally the major cost item. For example, in ethanol fuel produced by yeast fermentation, the cost of the sugar consumed by the yeast in the fermentation process represents the major production cost. As a result, biofuel producers looking to reduce their production costs are exploring the use of alternative low-cost feedstocks.
One biofuel-producing microorganism, which is particularly attractive in terms of both its ability to utilize a variety of inexpensive feedstocks and also to produce relatively large amounts of n-butanol, a very attractive biofuel that is much more similar to gasoline than is ethanol, is C. pasteurianum. C. pasteurianum is often found in whey isolates and it has a strong ability to utilize waste sugars generated during dairy processing. However, perhaps the most important inexpensive feedstock that C. pasteurianum utilizes very well is glycerine. Glycerine is produced as a major (i.e. 10%) by-product in biodiesel processing. At present, C. pasteurianum is the only known bacterium that can grow directly on glycerine as its sole carbon and energy source, while producing butanol as its major metabolite and product.
Molecular biology, and genetic engineering in particular, has, in some cases, emerged as a powerful approach for improving the biofuel production capacity, as well as feedstock utilization range, of industrial microorganisms. Today, there several major companies, such as Gevo, Butamax, and Cobalt Biofuels, that use genetically engineered organisms to produce biobutanol (that is, butanol produced from a biological process, as opposed to butanol produced by a chemical processing that does not involve living organisms) from a fermentation process. However, the ability to perform genetic engineering in a microorganism requires some basic tools such as: the ability to introduce new DNA into the microorganism, the ability to select for microorganisms carrying the new DNA, the ability to maintain the new DNA within the microorganism in a functional form (i.e. in a form that resists host defense and DNA degradation mechanisms), and the ability to express foreign genes within the microorganism. Prior to the present invention, such tools were lacking for C. pasteurianum; therefore C. pasteurianum was not amenable to genetic engineering and it has not previously been genetically engineered. In fact, out of the many species of Clostridial bacteria that have been found, only a very small number of them (approximately 5) have been developed for genetic engineering.
This invention discloses for the first time, the first successful genetic engineering of C. pasteurianum. This invention, together with the recently published genome of C. pasteurianum, provides the tools that will enable C. pasteurianum to be fully harnessed and developed for industrial butanol and biofuel production for the first time ever. Moreover, the tools that were developed to transform C. pasteurianum with foreign DNA will have broad applicability to enabling the transformation of bacteria that have not previously been genetically transformed.
SUMMARY OF THE INVENTIONThe present invention provides protocols that enable recombinant DNA constructs to be introduced into bacterial cells for which such protocols have not previously been disclosed. The present invention includes the bacterial cells containing recombinant DNA constructs.
In one preferred embodiment, the bacteria cells are of the anaerobic bacterium Clostridium pasteurianum. In another preferred embodiment, the invention describes one or more methyltransferases and their means of application, which are required to pretreat recombinant DNA constructs in order to enable their successful introduction into said bacterial cells. In another preferred embodiment, means and conditions are disclosed whereby said bacterial cells are rendered more amenable to transformation by recombinant DNA constructs by the application of one or more electrical pulses delivered to the bacterial cells while in the presence of the recombinant DNA constructs. In another preferred embodiments, information regarding antibiotic selection markers and recombinant DNA origins of replication is disclosed whereby one skilled in the art would be enabled to construct recombinant DNA constructs which can persist in the bacterial cells following transformation as independent genetic entities termed plasmids or which could enable such recombinant DNA constructs to become integrated into the bacterial genome under antibiotic selection pressure.
“Gene” refers to a nucleic acid sequence that encompasses a 5′ promoter region associated with the expression of the gene product, any intron and exon regions and 3′ or 5′ untranslated regions associated with the expression of the gene product.
“Transgene” refers to a nucleic acid sequence associated with the expression of a gene introduced into an organism. A transgene includes, but is not limited to, an endogenous gene or a gene not naturally occurring in the organism. A “transgenic organism” is any organism that stably incorporates a transgene in a manner that facilitates transmission of that transgene from the organism by any sexual or asexual method.
Tables
The invention consists of 2 parts:
1. Overcoming a CpaAI restriction enzyme within C. pasteurianum.
2. Overcoming the low electroporation transformation efficiency of C. pasteurianum.
A third important aspect of the invention is the development of DNA vectors and selection markers which enable the expression of foreign genes within C. pasteurianum.
1 Overcoming the CpaAI Restriction Enzyme within C. pasteurianum
Based on early genetic studies, it appears efforts were in place to conduct genetic manipulation of C. pasteurianum, since a method for producing and regenerating protoplasts (i.e. cells lacking cell walls) was developed (Clarke, et al., 1979) and a Type-II restriction endonuclease was identified as a potential barrier to gene transfer (Richards, et al., 1988). Successful conjugation-based plasmid transfer to C. pasteurianum has also been documented (Richards, et al., 1988), yet no protocol has been described, nor have any genetic mutants arisen from any prior work. Accordingly, no genetic tools are currently available for the manipulation of C. pasteurianum.
To develop a C. pasteurianum transformation protocol, we first assayed crude cell lysates for the presence of restriction-modification systems, which potently inhibit plasmid DNA transfer to bacteria. At least one Type-II restriction endonuclease, designated CpaAI with 5′-CGCG-3′ recognition and an isoschizomer of ThaI and FnuDII, has been previously identified in cell-free lysates of C. pasteurianum ATCC 6013 (Richards, et al., 1988). We initially prepared crude cell lysates through sonication of whole cells. As found in other species, such as C. acetobutylicum, lysates generated in this manner potently degraded all plasmid DNA substrates, presumably due to non-specific cell-wall-associated nucleases (data not shown). To overcome non-specific nuclease activity, we then aimed to assay CpaAI restriction activity using protoplast extracts, which allowed clear detection of CpaAI activity. Optimal digestion occurred between 2-4 hours incubation at 37° C. and produced a restriction pattern identical to that of BstUI, a commercial isoschizomer of CpaAI (
Initial Electrotransformation of C. pasteurianum
To electrotransform C. pasteurianum, we employed a series of E. coli-Clostridium shuttle vectors which differ only in their Gram-positive origins of replication: pMTL82151 (pBP1 on from C. botulinum); pMTL83151 (pCB102 on from C. butyricum); pMTL84151 (pCD6 on from C. difficile); and pMTL85141 (pIM13 on from Bacillus subtilis) (Heap, et al., 2009).
We utilized conditions common to clostridial electrotransformation procedures (Table 2) and M.FnuDII-methylated DNA. Of the four vectors tested, pMTL83151, pMTL84151, and pMTL85141 yielded colonies using thiamphenicol selection, corresponding to electrotransformation efficiencies of 0.7×101, 0.3×101, and 2.4×101 transformants μg−1 DNA, respectively. Accordingly, pMTL85141 was selected as the vector used for all subsequent electrotransformation work. Importantly, no transformants were obtained with unmethylated plasmid, validating the necessity to protect transforming DNA against the endogenous CpaAI restriction endonuclease. Interestingly, while in vivo methylation was essential for transformation, we did not obtain transformants when pMTL85141 was methylated in vitro with M.SssI or M.CviPI methyltransferases, although both enzymes protect pMTL85141 from digestion by CpaAI. This result is unexpected, and reinforces the degree of uncertainty and lack of obviousness of the choice of methylase which ultimately conferred successful C. pasteurianum transformation.
To confirm the presence of pMTL85141 in transformed colonies, we screened thiamphenicol-resistant colonies for the presence of the catP resistance marker within pMTL85141 using colony PCR (
2 Overcoming the Low Electroporation Transformation Efficiency of C. pasteurianum
The transformation efficiency obtained with electroporation of M.FnuDII-methylated pMTL85141 plasmid was 2.4×10 colonies per ug of DNA. This is a low transformation efficiency compared to efficiencies of up to 106 transformants per ug DNA obtained in other Clostridia. Such a low transformation efficiency would be problematic for applying some genetic engineering technology in C. pasteurianum, such as intron-mediated gene knockouts and homologous recombination-based gene editing, which often require the availability of abundant colonies for screening due to their low success rates. Therefore, we set out to develop a protocol which enabled high efficiency transformation. We systematically evaluated the effect on transformation efficiency of changing a number of parameters, which were based on modifying the integrity of the C. pasteurianum cell wall to permit easier entry of foreign DNA into the cell, and optimizing the electroporation conditions. These investigations are detailed below and together with the Examples, would enable one skilled in the art to transform C. pasteurianum at high efficiency.
(i) Cell-wall-weakening. We first investigated the use of cell-wall-weakening agents due to their potential to greatly enhance electrotransformation by weakening of the Gram-positive cell wall. A screening experiment was conducted to identify potential additives capable of enhancing electrotransformation of C. pasteurianum, including glycine, DL-threonine, lysozyme, and penicillin G (
As a result of the clear benefit of glycine on the electrotransformation efficiency, we set out to determine the optimum glycine regimen with respect to concentration and duration of exposure. This investigation was done concomitant with investigating the effect of sucrose on electrotransformation efficiency by providing osmoprotection during the various cell-wall-weakening glycine treatments. We tested glycine at 0.75, 1.0, and 1.25% in the presence of either 0.25 or 0.4 M sucrose, corresponding to nearly isotonic and hypertonic extracellular environments, respectively. The highest glycine concentration was selected as 1.25% to minimize growth inhibition, which becomes significant at concentrations equal to or greater than 1.5%. Increasing the sucrose concentration from 0.25 to 0.4 M led to a significant increase in electrotransformation efficiency under all glycine concentrations tested (
(ii) Osmoprotection. We continued to investigate the effect of the osmoprotectant concentration on electrotransformation efficiency during the subsequent washing and electroporation phase and the outgrowth phase following electroporation. Cells grown in the presence of 1.25% glycine and 0.4 M sucrose were washed and electroporated in the common clostridial SMP buffer containing either 0.27 M (isotonic) or 0.5 M (hypertonic) sucrose (
To assess the effect of sucrose osmoprotection during cell recovery immediately following delivery of the electric pulse, cells were grown, made electrocompetent, pulsed, and resuspended in 10 ml 2xYTG containing either 0.2 or 0.4 M sucrose (
Cell membrane solubilization. After developing a regimen to weaken the exterior cell wall while supporting cell viability with sucrose osmoprotection, we next sought to enhance transfer of plasmid DNA to C. pasteurianum with the use of ethanol to solubilize the cell membrane, a strategy which has proved effective with some species of Gram-negative bacteria. We also extended this approach to butanol, which elicits a more pronounced toxic effect on cells. To achieve maximum membrane solubilization without adversely affecting cell viability, we utilized concentrations near the toxicity threshold for many species of Clostridium, which were up to 15% (v/v) for ethanol and 2% (v/v) for butanol. Five minutes prior to electroporation, ethanol or butanol was added directly to the cell-DNA suspension. Ethanol added at 5 and 10% provided a 1.6- and 1.3-fold respective increase in electrotransformation efficiency, compared to the control experiment with no ethanol treatment (
Electric pulse parameters. We investigated the effects of the electrical pulse with respect to voltage (i.e., field strength), capacitance, and resistance (
DNA quantity and outgrowth duration. Finally, we evaluated the effect of DNA amount on both number of transformants and electrotransformation efficiency (
For assessing outgrowth duration, we incubated electroporated cells for 0, 2, 4, 6, or 16 hours prior to plating on selective medium. Growth in the form of gas formation and increased culture turbidity could be detected as early as 2 hours following transfer to recovery medium. Transformants could be obtained without recovery (i.e., 0 hours incubation), although at a significantly reduced efficiency (7.9- to 12.1-fold reduction compared to 2-16 hours incubation) (
Since many clostridial vectors favor the ermB determinant for erythromycin or clarithromycin selection, rather than catP-based thiamphenicol selection, we constructed pMTL85141ermB, a dual catP and ermB selectable plasmid. Comparable, high-level electrotransformation efficiencies (1.0-1.4×104 transformants μg−1 DNA) were obtained by selection of pMTL85141ermB using 15 μg/ml thiamphenicol, 4 μg/ml clarithromycin, or 20 μg/ml erythromycin. Control plasmid transformations lacking the ermB determinant failed to generate clarithromycin- or erythromycin-resistant colonies. Therefore, ermB-based clarithromycin or erythromycin selection is effective using C. pasteurianum.
To determine the generality of our high-efficiency electrotransformation protocol for other vectors, we also attempted electrotransfer of pSY6catP into C. pasteurianum. pSY6catP is a modified form of pSY6 (Shao, et al., 2007) whereby the ermB erythromycin-resistance determinant is replaced with catP from pMTL85141. pSY6 is one of several E. coli-Clostridium shuttle vectors (in addition to, e.g., the ClosTron system of vectors (Heap, et al., 2010)), which harbours the LI.ItrB group II intron machinery necessary for performing intron-mediated gene knockouts in clostridia. A pSY6-based vector was chosen because it possesses the same pIM13 replicon as pMTL85141, thereby eliminating potential variation in efficiency due to differences in the origin of replication. Unexpectedly, pSY6catP transformed C. pasteurianum at a significantly decreased efficiency of 1.1×101 transformants μg−1 DNA, an efficiency approximately 1.000-fold lower than achieved with pMTL85141. To rule out a vector size effect on the reduction in electrotransformation efficiency (pSY6catP is 8,498 bp, whereas pMTL85141 is 2,963 bp), we also attempted to transform pHT3, a 7,377 bp vector with the same fundamental vector components as pMTL85141ermB, in addition to a heterologous lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 (Tummala, et al., 1999) (Table 1). Unlike pSY6catP, pHT3 transformed at a high efficiency of 1.8×104 transformants μg−1 DNA, which is comparable to pMTL85141ermB. Therefore, the dramatic reduction in electrotransformation efficiency is likely not due to differences in plasmid size. At this point, we hypothesize the presence of an additional unidentified restriction system which targets certain common site(s) of pSY6catP, but not pMTL85141, pMTL85141ermB, or pHT3, much like the dcm-methylation-dependent restriction systems recently addressed in C. thermocellum and C. ljungdahlii. Our observation of the transformability of in-vivo-methylated plasmids, but not in-vitro-methylated plasmids, may also be the result of an unidentified methylation-dependent restriction system, which may or may not be the same one affecting pSY6catP. Nonetheless, even with the reduced electrotransformation efficiency of pSY6catP, we have used it to successfully introduce type II introns into the C. pasteurianum genome in preliminary experiments.
The unexpected result that there are some vectors, such as pSY6catP, which, even with M.FnuDII methylation, still fail to transform C. pasteurianum at high efficiency, emphasizes the value and lack of obviousness of our discovery of vectors such as pMTL85141, pMTL85141ermB, pHT3 that are capable of transforming C. pasteurianum at high efficiency once they are methylated in vivo by M.FnuDII.
In summary, we developed for the first time a high-efficiency transformation protocol for C. pasteurianum. Many variables needed to be carefully tuned to achieve optimal transformation efficiency, and there were several unexpected findings during the process of creating the invention. First, we determined that methylation was required to protect transformed plasmids from degradation by C. pasteurianum's CpaAI restriction enzyme system. However, surprisingly, not all methyltransferases which blocked CpaAI digestion activity in vitro were useful for protecting plasmids for transformation into C. pasteurianum. Only in vivo methylation by growth in a restriction deficient strain of E. coli, such as ER1821, harbouring the M.FnuDII methylation gene on a plasmid (in our case on plasmid pFnuDIIMKn), successfully protected plasmid for transformation into C. pasteurianum. At this point, we do not know whether the failure of the methylases M.SssI and M.CviPI to support transformation of C. pasteurianum was due to the methylases themselves or the use of the mythylases for in vitro, rather than in vivo, methylation.
Next, we sought to weaken the cell wall to try to increase transformation efficiency. Again, there were some unexpected results. While glycine, DL-threonine, lysozyme, and penicillin G have all been used for weakening the cell wall of gram-positive bacteria, in our experimental conditions, only glycine and DL-threonine significantly enhanced the level of electroporation. Importantly, it was also important to optimize the timing and concentration of glycine exposure to C. pasteurianum. Exposing the C. pasteurianum cells too early in their growth cycle, or with too great a concentration of glycine, led to a major loss of cell viability. The optimum glycine regimen for C. pasteurianum involved exposure of early exponential phase cells (at OD600 of 0.3-0.4) to 1.25% glycine for 2-3 hours. To our knowledge, this is the first use of glycine as a cell-wall weakening and electroporation-enhancing agent within the Clostridium genus.
As treatment with glycine compromised the cell wall, it became important to stabilize C. pasteurianum cells osmotically during the glycine treatment. However, here again, it was important to carefully monitor the time of application and concentration of the osmoprotectant agent. We discovered that the use of hypertonic 0.4 M sucrose in the growth medium significantly enhanced electrotransformation (
We also optimized the strength and duration of the electric field applied during electroporation. Generally, tailoring of the electric pulse was interdependent on the various optimizations of cell-wall weakening treatment; changing one required changing the other to maximize electrotransformation efficiency. We discovered that in contrast to the normal voltages of 2.0-2.5 kV (or 5.0-6.25 kV cm−1) which are used to transform other Clostridium bacteria, glycine-treated C. pasteurianum was found to benefit from a lower voltage of 1.8 kV (4.5 kV cm−1;
Once the cell wall is weakened, the foreign DNA must still cross through the cell membrane. Ethanol is known as a membrane-solubilizing agent that enhances the transformation of Escherichia coli. However, it has not previously been used to improve electrotransformation of Clostridia. We discovered that adding 5-10% ethanol to the electroporation mixture had a clear positive effect on electrotransformation (
Taken together, with the optimization of various electroporation parameters, and the careful timed additions of specific concentrations of cell wall and cell membrane modifying agents, we achieved a 3.000-fold increase in electroporation efficiency compared to our initial electrotransformation attempt using common clostridial electroporation conditions (Table 3). Our final maximum transformation efficiency was 7.5×104 tranformants ug−1 DNA, which is among the highest reported in the Clostridium genus. This transformation efficiency is sufficiently high to perform all of the normal genetic engineering manipulations needed to produce high-producing industrial strains.
Finally, in our invention, we disclosed for the first time, a number of vectors and selection markers, which are effective for transforming C. pasteurianum. These new tools included vectors with the pCB102 origin of replication from C. butyricum (pMTL83151), pCD6 origin of replication from C. difficile (pMTL84151), and the pIM13 from B. subtilis (pMTL85141, pMTL85141ermB, and pHT3) and vectors carrying catP and ermB genes for thiamphenicol and erythromycin/clarithromycin selection, respectively. We established that concentrations of 10-15 μg/ml thiamphenicol, 4 μg/ml clarithromycin, and 20 μg/ml erythromycin were appropriate for selection of transformed colonies. Importantly, not all vectors that we tried to transform were equally effective in generating transformation colonies in C. pasteurianum, in spite of being adequately protected from CpaAI degradation by methylation. In particular, the class of pSY6 vectors, which are conventionally used to transform group II intron-mediated gene knockout machinery into clostridial cells (Shao, et al., 2007), did not transform efficiently with our protocol. This implies that the vectors that we identified as supporting high-efficiency transformation were not obvious since we could not predict beforehand which vectors would and would not transform C. pasteurianum efficiently.
Nonetheless, even with the pSY6 vectors that transformed C. pasteurianum poorly, we were able to generate sufficient colonies following transformation to detect successful gene knockout events.
Below, further examples are provided of the detailed use of the invention to achieve successful transformation of C. pasteurianum.
EXAMPLESThe following examples are provided by way of illustration and not by limitation.
Example 1The bacterial strains, plasmids, and oligonucleotides utilized in this invention are listed in Table 1. E. coli DH5α was utilized for routine vector construction and propagation, and E. coli ER1821 for maintenance of M.FnuDII-methylated E. coli-C. pasteurianum shuttle vectors. C. pasteurianum ATCC™ 6013 (Winogradsky 5; W5) was acquired from the American Type Culture Collection (Manassas, Va., USA). Modular pMTL-series shuttle vectors (Heap, et al., 2009) were kindly provided by Prof. Nigel Minton (University of Nottingham, Nottingham, UK). Plasmids pFnuDIIM (Lunnen, et al., 1988), pSC12 (Zhao, et al., 2003), and pSY6 (Shao, et al., 2007) were respectively provided by Dr. Geoffrey Wilson (New England Biolabs, Inc. (NEB), Ipswich, Mass., USA), Prof. George Bennett (Rice University, Houston, Tex., USA), and Prof. Sheng Yang (Shanghai Institutes for Biological Sciences, Shanghai, China). Plasmids pHT3 (Tummala, et al., 1999) and pIMP1 (Mermelstein, et al., 1992) were provided by Prof. Terry Papoutsakis (University of Delaware, Newark, Del., USA). Oligonucleotide primers were synthesized and purified by Integrated DNA Technologies (IDT; Iowa City, Iowa, USA) using standard desalting.
Bacteria Growth and MaintenanceUnless stated otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA) and stock solutions were prepared according to the manufacturer's recommendations. E. coli strains were grown aerobically at 37° C. in lysogeny broth (LB; 10 g/l NaCl, 5 g/l Bacto yeast extract, and 10 g/l Bacto tryptone). Solid and liquid cultures of recombinant E. coli were supplemented with 100, 34, or 30 μg/ml of ampicillin, chloramphenicol, and kanamycin, respectively. For selection of strains harboring two compatible plasmids, antibiotic concentrations were reduced by 50%. Recombinant E. coli stocks were stored at −80° C. in 15% glycerol. Unless specified otherwise, growth and manipulation of C. pasteurianum was performed in a controlled anaerobic atmosphere (85% N2, 10% H2, and 5% CO2) within an anaerobic chamber (Plas-Labs, Inc.; Lansing, Mich., USA). Oxygen was purged from growth medium by autoclaving and trace O2 was reduced using a palladium catalyst fixed to the heating unit of the anaerobic chamber. Agar-solidified medium was prepared aerobically and allowed to equilibrate within the anaerobic chamber for at least 36 hours prior to use. Anaerobic conditions were monitored by addition of 1 mg/l resazurin to both solid and liquid media. Solid and liquid cultures of recombinant C. pasteurianum were supplemented with 15 μg/ml thiamphenicol. Cells were maintained as spores on solidified 2xYTG (16 g/l Bacto tryptone, 10 g/l Bacto yeast extract, 5 g/l glucose, 5 g/l NaCl, and 12 g/l agar) plates. Sporulated agar plate stocks were prepared by streaking colonies from an exponential-phase culture (OD600 of 0.4-0.6) and cultivating for more than seven days under anaerobic conditions, followed by exposure and storage in air at 4° C. for up to two months. For long-term storage, vegetative stock cultures (OD600 of 0.4-0.6) were prepared and stored at −80° C. in 10% glycerol by inoculating a single sporulated plate colony into 10 ml 2xYTG and heat shocking at 80° C. for 10 minutes to induce germination.
DNA Isolation and ManipulationPlasmid DNA was extracted and purified from E. coli DH5α and ER1821 using an EZ-10 Spin Column Plasmid DNA Miniprep Kit from Bio Basic, Inc. (Markham, ON, Canada). Recombinant DNA manipulations were performed according to standard procedures (Sambrook, et al., 1989). Taq DNA polymerase, restriction endonucleases, CpG (M.SssI) and GpC (M.CviPI) methyltransferases, Quick Ligation Kit, and 1 kb DNA ladder were purchased from NEB (Ipswich, Mass., USA). Pfu DNA polymerase and RNase A were purchased from Bio Basic, Inc. (Markham, ON, Canada). All commercial enzymes and kits were used according to the manufacturer's instructions.
Colony PCR of wild-type and recombinant C. pasteurianum was performed by suspending single colonies in 50 μl colony lysis buffer (20 mM Tris-HCl, pH 8.0, containing 2 mM EDTA and 1% Triton X-100), heating in a microwave for 2 minutes at maximum power setting, and adding 1 μl of the resulting cell suspension to a 9 μl PCR containing Standard Taq DNA Polymerase (NEB; Ipswich, Mass., USA). An initial denaturation of 5 minutes at 95° C. was employed to further cell lysis. Colonies screened in this manner by suspension in deionized H2O failed to yield appreciable amplification.
Vector construction
Plasmid pFnuDIIMKn was derived from pFnuDIIM to allow methylation of E. coli-C. pasteurianum shuttle vectors and possesses a kanamycin-resistance determinant, as both pFnuDIIM (Lunnen, et al., 1988) and the E. coli-C. pasteurianum shuttle vectors used in this study carry the same chloramphenicol-resistance marker. First, an FRT-kan-FRT PCR cassette was amplified from plasmid pKD4 (Datsenko and Wanner, 2000) using primers KnFRT.BlpI.S (SEQ ID NO: 1) and KnFRT.XhoI.AS (SEQ ID NO: 2) and inserted into the MCS of BlpI/XhoI-digested pET-20b(+) (Novagen; Madison, Wis., USA) to generate pETKnFRT. Next, the FRT-kan-FRT cassette was digested out of pETKnFRT using ScaI and EcoRI and subcloned into the corresponding restriction sites within the catP gene of pFnuDIIM to yield pFnuDIIMKn.
Plasmid pSY6catP was derived from pSY6 (Shao, et al., 2007) by swapping the ermB marker with the catP determinant from pSC12 (Zhao, et al., 2003). The internal BsrGI recognition site within the coding sequence of catP was mutated by introducing two silent mutations using splicing by overlap extension (SOE) PCR to prevent interference with future group II intron retargeting, which requires use of BsrGI. The catP gene was amplified in two parts from template pSC12 using primer sets catP.BcII.S (SEQ ID NO: 5)/pSC12.SOE.AS (SEQ ID NO: 6) and pSC12.SOE.S (SEQ ID NO: 7)/catP.ClaI.AS (SEQ ID NO: 8) with 22 bp of overlap between products. The resulting overlapping PCR products were separated on a 2.0% agarose gel, pierced three times with a P10 micropipette tip, and used as template in a SOE PCR by cycling for 10 cycles prior to adding primers catP.BcII.S (SEQ ID NO: 5) and catP.ClaI.AS (SEQ ID NO: 8) and cycling for 25 additional cycles. The mutated PCR product was purified using a EZ-10 Spin Column PCR Products Purification Kit (Bio Basic, Markham, ON, Canada), digested with BclI/ClaI, and inserted into the corresponding sites of pSY6 to generate pSY6catP.
Plasmid pMTL85141ermB was derived from pMTL85141 via insertion of the ermB marker from pIMP1 into pMTL85141. The ermB gene and associated promoter was PCR-amplified from template pIMP1 using primers ermB.NdeI.S (SEQ ID NO: 3) and ermB.Pvul.AS (SEQ ID NO: 4). The resulting 1,238 bp PCR product was purified using an EZ-10 Spin Column PCR Products Purification Kit (Bio Basic, Markham, ON, Canada), digested with NdeI/PvuI, and inserted into the corresponding sites of pMTL85141 to generate pMTL85141ermB.
Preparation of Electrocompetent Cells and ElectrotransformationFor preparation of electrocompetent cells of C. pasteurianum using the high-level protocol, a seed culture was first prepared by inoculating 20 ml of reduced 2xYTG with 0.2 ml of a thawed glycerol stock. The culture was then 20−2-diluted and, following overnight growth at 37° C., 1 ml of the seed culture was transferred to a 125 ml Erlenmeyer flask containing 20 ml of reduced 2xYTG. Cells were grown to early exponential phase (OD600 of 0.3-0.4), at which time filter-sterilized stock solutions of 2 M sucrose and 18.77% glycine were added to respective concentrations of 0.4 M and 1.25%. Growth was resumed until the culture attained an OD600 of 0.6-0.8 (approximately 2-3 h) and 20 ml culture was transferred to a 50 ml pre-chilled, screw-cap centrifuge tube. At this point, all manipulations were performed at 4° C. using an ice-bath and pre-chilled reagents. Cells were removed from the anaerobic chamber and collected by centrifugation at 8,500×g and 4° C. for 20 minutes. The resulting cell pellet was returned to the anaerobic chamber and washed once in 5 ml of filter-sterilized SMP buffer (270 mM sucrose, 1 mM MgCl2, and 5 mM sodium phosphate, pH 6.5). Following centrifugation, the final cell pellet was resuspended in 0.6 ml SMP buffer.
For transfer of plasmids to C. pasteurianum, E. coli-C. pasteurianum shuttle vectors were first co-transformed with pFnuDIIMKn into E. coli ER1821 to methylate the external cytosine residue within 5′-CGCG-3′ tetranucleotide recognition sites of CpaAI. Plasmid mixtures were then isolated and 0.5 μg, suspended in 20 μl of 2 mM Tris-HCl, pH 8.0, was added to 580 μl of C. pasteurianum electrocompetent cells. The cell-DNA mixture was transferred to a pre-chilled electroporation cuvette with 0.4 cm gap (Bio-Rad; Richmond, Calif., USA), 30 μl of cold 96% ethanol was added, and the suspension was incubated on ice for 5 minutes. A single exponential decay pulse was applied using a Gene Pulser (Bio-Rad, Richmond, Calif., USA) set at 1.8 kV, 25 μF, and ∞Ω, generating a time constant of 12-14 ms. Immediately following pulse delivery, the cuvette was flooded with 1 ml 2xYTG medium containing 0.2 M sucrose and the entire suspension was transferred to 9 ml of the same medium. Recovery cultures were incubated for 4-6 hours prior to plating 50-250 μl aliquots onto 2xYTG agar plates containing 15 μg/ml thiamphenicol, 4 μg/ml clarithromycin, or 20 μg/ml erythromycin. Plates were incubated for 2-4 days under secondary containment within 3.4 L Anaerobic Jars each equipped with a 3.5 L Anaerobic Gas Generating sachet (Oxoid Thermo Fisher; Nepean, ON, Canada).
Claims
1. A method for introducing recombinant DNA constructs into one or more bacterium.
2. The method of claim 1 wherein said bacterium is a gram-positive bacteria.
3. The method of claim 1 wherein said bacterium belongs to the genus Clostridia.
4. The method of claim 1 wherein said bacterium is Clostridium pasteurianum.
5. The method of claim 1 wherein said method involves the delivery of one or more electrical pulses to said bacterium.
6. The method of claim 1 wherein said method involves the use of methylation to block the activity of a restriction enzyme within said bacterium.
7. The method of claim 1 wherein said method involves the use of a methylase which methylates a cytosine residue within the deoxyribonucleotide sequence 5′-cytosine-guanine-cytosine-guanine-3′.
8. The method of claim 1 wherein said method involves the use of a cell-wall weakening agent.
9. The method of claim 8 wherein said cell-wall weakening agent is selected from the group consisting of glycine and DL-threonine.
10. The method of claim 9 wherein said method involves the use of an osmoprotectant agent.
11. The method of claim 10 wherein said osmoprotectant is selected from the group consisting of sucrose, lactose, sorbitol, and mannitol.
12. The method of claim 9 where said method involves the use of ethanol.
13. The method of claim 4 where said recombinant DNA construct contains an origin of replication selected from the group consisting of pCB102 from Clostridium butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis.
14. The method of claim 4 wherein said recombinant DNA construct contains a DNA sequence which encodes an enzyme that confers resistance to an antibiotic selected from the group consisting of thiamphenicol, clarithromycin, and erythromycin.
15. The method of claim 4 wherein said method comprises:
- (i) the delivery of one or more electrical pulses to said bacterium.
- (ii) the use of one or more cell-wall weakening agents selected from the group comprising glycine and DL-threonine.
- (iii) the use of one or more osmoprotectants selected from the group consisting of sucrose, lactose, mannitol, and sorbitol.
16. A bacterial cell which contains one or more recombinant DNA constructs.
17. The bacterial cell of claim 16 wherein said bacterial cell is from a gram-positive bacteria.
18. The bacterial cell of claim 16 wherein said bacterial cell is from a bacteria that is a member of the genus Clostridium.
19. The bacteria cell of claim 16 wherein said bacterial cell is from Clostridium pasteurianum.
20. The bacterial cell of claim 16 wherein said one or more recombinant DNA constructs were introduced into said bacterial cell by a method involving the delivery of one or more electrical pulses to said bacterium.
21. The bacterial cell of claim 16 wherein said one or more recombinant DNA constructs were introduced into said bacterial cell by a method involving the use of methylation to block the activity of a restriction enzyme within said bacterium.
22. The bacterial cell of claim 16 wherein said one or more recombinant DNA constructs were introduced into said bacterial cell by a method involving the use of a methylase which methylates a cytosine residue within the deoxyribonucleotide sequence 5′-cytosine-guanine-cytosine-guanine-3′.
23. The bacterial cell of claim 16 wherein said one or more recombinant DNA constructs were introduced into said bacterial cell by a method involving the use of a cell-wall weakening agent.
24. The bacterial cell of claim 23 wherein said cell-wall weakening agent is selected from the group consisting of glycine and DL-threonine.
25. The bacterial cell of claim 24 wherein said method involves the use of an osmoprotectant agent.
26. The bacterial cell of claim 25 wherein said osmoprotectant is selected from the group consisting of sucrose, lactose, sorbitol, and mannitol.
27. The bacterial cell of claim 24 where said method involves the use of ethanol.
28. The bacterial cell of claim 19 where said one or more recombinant DNA constructs contain an origin of replication selected from the group consisting of pCB102 from Clostridium butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis.
29. The bacterial cell of claim 19 wherein said one or more recombinant DNA constructs contain a DNA sequence which encodes an enzyme that confers resistance to an antibiotic selected from the group consisting of thiamphenicol, clarithromycin, and erythromycin.
30. The bacterial cell of claim 19 wherein said one or more recombinant DNA constructs are introduced into said bacterial cell by a method comprising:
- (i) the delivery of one or more electrical pulses to said bacterium.
- (ii) the use of one or more cell-wall weakening agents selected from the group comprising glycine and DL-threonine.
- (iii) the use of one or more osmoprotectants selected from the group consisting of sucrose, lactose, mannitol, and sorbitol.
Type: Application
Filed: Jan 8, 2014
Publication Date: Jul 10, 2014
Applicant: Algaeneers Inc. (Hamilton)
Inventors: Michael Evan Pyne (Hamilton), Murray Moo Young (Waterloo), Duane Andrew Chung (Mississauga), Chih-Hsiung Perry Chou (Waterloo)
Application Number: 14/150,764
International Classification: C12N 15/74 (20060101);